This research program targets at the search for the true endogenous ligands for the iNKT cells development. Conventional CD4+ and CD8+ T cells of the immune system recognize specific peptide antigens bound to major histocompatibility complex (MHC) class II or MHC class I molecules, respectively. In contrast, a specialized subpopulation of T cells called invariant natural killer T (iNKT) cells recognizes glycolipid antigens presented by the MHC class I-like molecule, CD1d. Upon stimulation, iNKT cells produce large amounts of T helper 1 (Th1), Th2 cytokines and chemokines in a rapid burst which has a broad immunological influence, including protection against autoimmune diseases, host responses to parasites and bacteria, and anti-tumor responses. Although the well-known a-GalCer can dramatically stimulate the iNKT cells, it's an exogenous antigen isolated from the marine sponge. During the development of iNKT cells in the thymus from the T cell precursor, an endogenous antigen is necessary for the process of being presented by CD1d to the randomly generated TCR. iGb3 was reported as a possible endogenous antigen in both mice and human. However, studies from independent laboratories have challenged the hypothesis that iGb3 is the primary ligand responsible for iNKT cell development in mice. Specially, no iGb3 was detected in either the mice or human thymus by NP-HPLC analysis. The iGb3 synthase knockout (iGb3S-/-) mice furthermore showed normal iNKT cells numbers in the thymus, spleen, and liver. Further experiments showed that there is no iGb3S identified in any human tissue by analysis of the iGb3S mRNA, and the chimeric molecules containing the catalytic domain of human iGb3S did not show any functional activity in synthesis of iGb3 due to at least one amino acid substitution. Despite the dispute of the existence of iGb3 in mice and human thymus samples or other tissues, the activity of iGb3 in the stimulation of iNKT cells is well accepted. This provides us with important clues in the search for the genuine iNKT cell ligands inside the thymus for iNKT cell development. Accordingly, in this proposal, we hypothesize one type of glycolipids as the potential primary ligands based on the biosynthesis and degradation pathway of glycolipids in mouse and human, on the crystal structures of CD1d/1-GalCer/TCR and CD1d/iGb3 complexes, as well as on the three dimensional structure of iGb3. The possible antigens are three blood type glycosylceramides which possess structures similar to that of iGb3 and have the corresponding synthetic pathway in both mice and humans. All of these ligands possess a fucose moiety on the C2 position of a galactose. The computational docking of the blood B type glycosylceramide shows that this compound can be accommodated in the cavity between TCR and CD1d. Although these blood type glycosylceramides are much less studied so far, two of them have been identified in pig tissues, which implies that they may also exist in mouse and human tissues, such as thymus and spleen. Three specific aims are proposed in this program: 1. Preparation of three different blood type glycoceramides which include Fuc1(1,2)Gal2(1,4)Glc2(1,1)ceramide (blood O type), GalNAc1(1,3)[Fuc1(1,2)]Gal2(1,4)Glc2(1,1)ceramide (blood A type) and Gal1(1,3)[Fuc1(1,2)]Gal2(1,4)Glc2(1,1)ceramide (blood B type). The metabolic stable analogs with thio-atom as linkage between the saccharides will also be prepared. 2. Evaluate the biological activity of these blood type glycosylceramides and metabolic analogs with different models. Evaluate the effects of blood type glycosylceramides on iNKT cells development. 3. Identify the potential blood type glycosylceramide antigens in mice tissues. iNKT cells serve as the bridge between innate and adaptive immune system. Identification of endogenous ligand of iNKT cells will benefit the understanding of the mechanism of iNKT cells developing and proliferation, and its relationship with other immune cells, such as T cells, B cells, and macrophage cells. This will also help researchers to develop methods to apply the principle into clinic application. PUBLIC HEALTH RELEVANCE: iNKT cells are a newly discovered sub-population of T cells. Upon stimulation with glycolipids, iNKT cells rapidly release a large amount of cytokines which is very important in the inflammatory response and suppressing the tumor proliferation. iNKT cells serve as the bridge between innate and adaptive immune system. Identification of endogenous ligand of iNKT cells will benefit the understanding of the mechanism of iNKT cells developing and proliferation, and its relationship with other immune cells, such as T cells, B cells, and macrophage cells. This will also help researchers to develop methods to apply the principle into clinic application.